The major goal of this project is to identify an optimal adenovirus- based vaccination strategy against homologue (mEGP) of the human colorectal carcinoma-associated GA733 antigen. Since mice express mEGP on normal tissues (similar to GA733 Ag in humans), this animal model allows testing of the vaccine in an immunologically tolerant host and evaluation of potential toxicity. These studies will optimize the ability of this vaccine to overcome tolerance to this tumor antigen in settings that closely mimic patients with colon cancer. Therefore, they will have an impact on the initial human clinical trial (see Project 3, Aim 2) and will provide the basis for the testing of improved human colon cancer vaccines in the near future. We have prepared an adenovirus-based mEGP vaccine and have already shown that it can induce tumor protection in the mouse when used in combination with interleukin-2 (IL-2). This is the basis for our proposed series of experiments. Our initial experiments will address issues of immediate clinical relevance, i.e., administration of the vaccine by a cutaneous route, the need for one versus two doses of the vaccine, and the appropriate use of cytokine. Having identified parameters that limit or enhance our vaccine in a subcutaneous tumor model, we will than address whether our vaccination strategy is effective against more advanced and metastatic disease. This will provide us with the opportunity to improve our vaccine by studying the effects of different booster strategies, the use of other cytokines and the use of functionally active fragments (from Project 1) of mEGP. Having optimized our vaccine, we will study its mechanism of action by defining the target cells through which the adenovirus elicits anti-mEGP immunity, the role of the adenoviral proteins in the induction of the immune response, and the cell types that contribute to the effectiveness of the immune response.